微流控芯片操纵传输及实时监测单细胞量子释放

微流控芯片技术用于细胞生化分析已引起了广泛关注.Harrison等首次在微流控芯片上对细胞群体进行操纵、传输及反应.yang等在微流控芯片上操纵细胞群体的排列,并用荧光检测细胞群体摄取钙的反应.至今还未见到微流控芯片对单个细胞进行操纵传输、定位及实时监测的报道.单细胞受激释放的监测对探索生物体神经传导具有重要意义.     

T cell chemotaxis in a simple microfluidic device

This paper describes the use of a simple microfluidic device for studying T cell chemotaxis. The microfluidic device is fabricated in poly(dimethylsiloxane) (PDMS) using soft-lithography and consists of a "Y" type fluidic channel. Solutions are infused into the device by syringe pumps and generate a concentration gradient in the channel by diffusion. We show that the experimentally measured gradient profiles agree nicely with theoretical predictions and the gradient is stable in the observation region for cell migration. Using this device, we demonstrate robust chemotaxis of human T cells in response to single and competing gradients of chemokine CCL19 and CXCL12. Because of the simplicity of the device, it can flexibly control gradient generation in space and time, and would allow generation of multiple gradient conditions in a single chip for highly parallel chemotaxis experimentation. Visualization of T cell chemotaxis has previously been limited to studies in 3D matrices or under agarose assays, which do not allow precise control or variation in conditions. Acknowledging the importance of lymphocyte homing in the adaptive immune response, the ability to study T cell chemotaxis in microfluidic devices offers a new approach for investigating lymphocyte migration and chemotaxis in vitro.     

A microfluidic system to study the cytotoxic effect of drugs: the combined effect of celecoxib and 5-fluorouracil on normal and cancer cells

We have investigated the response of normal and cancer cells to exposure a combination of celecoxib (Celbx) and 5-fluorouracil (5-FU) using a lab-on-a-chip microfluidic device. Specifically, we have tested the cytotoxic effect of Celbx on normal mouse embryo cells (Balb/c 3T3) and human lung carcinoma cells (A549). The single drugs or their combinations were adjusted to five different concentrations using a concentration gradient generator (CGG) in a single step. The results suggest that Celbx can enhanced the anticancer activity of 5-FU by stronger inhibition of cancer cell growth. We also show that the A549 cancer cells are more sensitive to Celbx than the Balb/c 3T3 normal cells. The results obtained with the microfluidic system were compared to those obtained with a macroscale in vitro cell culture method. In our opinion, the microfluidic system represents a unique approach for an evaluation of cellular response to multidrug exposure that also is more simple than respective microwell plate assays.

High-throughput tracking of single yeast cells in a microfluidic imaging matrix

Time-lapse live cell imaging is a powerful tool for studying signaling network dynamics and complexity and is uniquely suited to single cell studies of response dynamics, noise, and heritable differences. Although conventional imaging formats have the temporal and spatial resolution needed for such studies, they do not provide the simultaneous advantages of cell tracking, experimental throughput, and precise chemical control. This is particularly problematic for system-level studies using non-adherent model organisms such as yeast, where the motion of cells complicates tracking and where large-scale analysis under a variety of genetic and chemical perturbations is desired. We present here a high-throughput microfluidic imaging system capable of tracking single cells over multiple generations in 128 simultaneous experiments with programmable and precise chemical control. High-resolution imaging and robust cell tracking are achieved through immobilization of yeast cells using a combination of mechanical clamping and polymerization in an agarose gel. The channel and valve architecture of our device allows for the formation of a matrix of 128 integrated agarose gel pads, each allowing for an independent imaging experiment with fully programmable medium exchange via diffusion. We demonstrate our system in the combinatorial and quantitative analysis of the yeast pheromone signaling response across 8 genotypes and 16 conditions, and show that lineage-dependent effects contribute to observed variability at stimulation conditions near the critical threshold for cellular decision making.     

Flow characterization of a microfluidic device to selectively and reliably apply reagents to a cellular network

A three-dimensional microfluidic device has been successfully fabricated and the flow streams characterized for eventual use in studying communication in an in vitro network of nerve cells. The microfluidic system is composed of two layers of channels: a lower layer for the delivery of pharmacological solutions and an upper layer of channels used to direct the flow of the pharmacological solution streams and perfuse the cells with media and nutrients. Flow profiles have been characterized with computational fluid dynamics simulations, confocal fluorescence microscopy, and carbon-fiber amperometry, which have been used to map changes in flow profiles at different bulk flow rates. Ultimately, the microfluidic system and incorporated cell network will show how networked neurons adapt, compensate, and recover after being exposed to different chemical compounds.     

Droplet microfluidics with magnetic beads: a new tool to investigate drug–protein interactions

In this study, we give the proof of concept for a method to determine binding constants of compounds in solution. By implementing a technique based on magnetic beads with a microfluidic device for segmented flow generation, we demonstrate, for individual droplets, fast, robust and complete separation of the magnetic beads. The beads are used as a carrier for one binding partner and hence, any bound molecule is separated likewise, while the segmentation into small microdroplets ensures fast mixing, and opens future prospects for droplet-wise analysis of drug candidate libraries. We employ the method for characterization of drug–protein binding, here warfarin to human serum albumin. The approach lays the basis for a microfluidic droplet-based screening device aimed at investigating the interactions of drugs with specific targets including enzymes and cells. Furthermore, the continuous method could be employed for various applications, such as binding assays, kinetic studies, and single cell analysis, in which rapid removal of a reactive component is required.     

Monitoring spatial distribution of ethanol in microfluidic channels by using a thin layer of cholesteric liquid crystal

Monitoring spatial distribution of chemicals in microfluidic devices by using traditional sensors is a challenging task. In this paper, we report utilization of a thin layer of cholesteric liquid crystal for monitoring ethanol inside microfluidic channels. This thin layer can be either a polymer dispersed cholesteric liquid crystal (PDCLC) layer or a free cholesteric liquid crystal (CLC) layer separated from the microfluidic device by using a thin film of PDMS. They both show visible colorimetric responses to 4% of ethanol solution inside the microfluidic channels. Moreover, the spatial distribution of ethanol inside the microfluidic channel can be reflected as a color map on the CLC sensing layers. By using this device, we successfully detected ethanol produced from fermentation taking place inside the microfluidic channel. These microfluidic channels with embedded PDCLC or embedded CLC offer a new sensing solution for monitoring volatile organic compounds in microfluidic devices.     

Selective trapping of single mammalian breast cancer cells by insulator-based dielectrophoresis

The trapping or immobilization of individual cells at specific locations in microfluidic platforms is essential for single cell studies, especially those requiring cell stimulation and downstream analysis of cellular content. Selectivity for individual cell types is required when mixtures of cells are analyzed in heterogeneous and complex matrices, such as the selection of metastatic cells within blood samples. Here, we demonstrate a microfluidic device based on direct current (DC) insulator-based dielectrophoresis (iDEP) for selective trapping of single MCF-7 breast cancer cells from mixtures with both mammalian peripheral blood mononuclear cells (PBMC) as well MDA-MB-231 as a second breast cancer cell type. The microfluidic device has a teardrop iDEP design optimized for the selective capture of single cells based on their differential DEP behavior under DC conditions. Numerical simulations adapted to experimental device geometries and buffer conditions predicted the trapping condition in which the dielectrophoretic force overcomes electrokinetic forces for MCF-7 cells, whereas PBMCs were not trapped. Experimentally, selective trapping of viable MCF-7 cells in mixtures with PBMCs was demonstrated in good agreement with simulations. A similar approach was also executed to demonstrate the selective trapping of MCF-7 cells in a mixture with MDA-MB-231 cells, indicating the selectivity of the device for weakly invasive and highly invasive breast cancer cells. The DEP studies were complemented with cell viability tests indicating acceptable cell viability over the course of an iDEP trapping experiment.

Integrated microfluidic array plate (iMAP) for cellular and molecular analysis

Just as the Petri dish has been invaluable to the evolution of biomedical science in the last 100 years, microfluidic cell assay platforms have the potential to change significantly the way modern biology and clinical science are performed. However, an evolutionary process of creating an efficient microfluidic array for many different bioassays is necessary. Specifically for a complete view of a cell response it is essential to incorporate cytotoxic, protein and gene analysis on a single system. Here we present a novel cellular and molecular analysis platform, which allows access to gene expression, protein immunoassay, and cytotoxicity information in parallel. It is realized by an integrated microfluidic array plate (iMAP). The iMAP enables sample processing of cells, perfusion based cell culture, effective perturbation of biologic molecules or drugs, and simultaneous, real-time optical analysis for different bioassays. The key features of the iMAP design are the interface of on-board gravity driven flow, the open access input fluid exchange and the highly efficient sedimentation based cell capture mechanism (～100% capture rates). The operation of the device is straightforward (tube and pump free) and capable of handling dilute samples (5-cells per experiment), low reagent volumes (50 nL per reaction), and performing single cell protein and gene expression measurements. We believe that the unique low cell number and triple analysis capabilities of the iMAP platform can enable novel dynamic studies of scarce cells.     

A self-loading microfluidic device for determining the minimum inhibitory concentration of antibiotics

This article describes a portable microfluidic technology for determining the minimum inhibitory concentration (MIC) of antibiotics against bacteria. The microfluidic platform consists of a set of chambers molded in poly(dimethylsiloxane) (PDMS) that are preloaded with antibiotic, dried, and reversibly sealed to a second layer of PDMS containing channels that connect the chambers. The assembled device is degassed via vacuum prior to its use, and the absorption of gas by PDMS provides the mechanism for actuating and metering the flow of fluid in the microfluidic channels and chambers. During the operation of the device, degas driven flow introduces a suspension of bacterial cells, dissolves the antibiotic, and isolates cells in individual chambers without cross contamination. The growth of bacteria in the chambers in the presence of a pH indicator produces a colorimetric change that can be detected visually using ambient light. Using this device we measured the MIC of vancomycin, tetracycline, and kanamycin against Enterococcus faecalis 1131, Proteus mirabilis HI4320, Klebsiella pneumoniae, and Escherichia coli MG1655 and report values that are comparable to standard liquid broth dilution measurements. The device provides a simple method for MIC determination of individual antibiotics against human pathogens that will have applications for clinical and point-of-care medicine. Importantly, this device is designed around simplicity: it requires a single pipetting step to introduce the sample, no additional components or external equipment for its operation, and provides a straightforward visual measurement of cell growth. As the device introduces a novel approach for filling and isolating dead-end microfluidic chambers that does not require valves and actuators, this technology should find applications in other portable assays and devices.     

Cytotoxicity analysis of water disinfection byproducts with a micro-pillar microfluidic device

Water disinfection byproducts (DBPs) are a class of chemicals that are produced when chemical disinfectants react with organic materials in untreated water. Cytotoxicity and genotoxicity of DBPs have been systematically evaluated to compile a comparative, quantitative database of in vitro mammalian cell toxicity of DBPs. However, one of the most challenging limitations for current DBP cytotoxicity assessment assays is sample availability. Although our current cytotoxicity assay using a 96-well microplate has been designed to reduce sample consumption, further minimization of the size of the test system would allow us to explore various possibilities for point-of-care applications. We have developed a microfluidic device with micro-pillars that shows high uniformity in distribution of cells across all chambers with low cell count. We compare the performance between the 96-well microplate and the microfluidic device by running 72-hour standalone-on-chip cell culture and cytotoxicity analysis experiments, using dimethyl sulfoxide (DMSO) and ethanol as model toxic agents, and bromoacetic acid (BAA) as a representative DBP. The results show close agreement between the two systems. The measured LC(50) values for the 96-well microplate and the microfluidic device are 1.54% v/v and 1.27% v/v for DMSO, 1.44% v/v and 2.92% v/v for ethanol, and 17.6 μM and 8.20 μM for BAA, respectively. The micro-pillar microfluidic device offers a great reduction in sample consumption while maintaining the accuracy of the cytotoxicity analyses of water disinfection byproducts.     

Label-free quantitation of peptide release from neurons in a microfluidic device with mass spectrometry imaging

Microfluidic technology allows the manipulation of mass-limited samples and when used with cultured cells, enables control of the extracellular microenvironment, making it well suited for studying neurons and their response to environmental perturbations. While matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) provides for off-line coupling to microfluidic devices for characterizing small-volume extracellular releasates, performing quantitative studies with MALDI is challenging. Here we describe a label-free absolute quantitation approach for microfluidic devices. We optimize device fabrication to prevent analyte losses before measurement and then incorporate a substrate that collects the analytes as they flow through a collection channel. Following collection, the channel is interrogated using MS imaging. Rather than quantifying the sample present via MS peak height, the length of the channel containing appreciable analyte signal is used as a measure of analyte amount. A linear relationship between peptide amount and band length is suggested by modeling the adsorption process and this relationship is validated using two neuropeptides, acidic peptide (AP) and α-bag cell peptide [1-9] (αBCP). The variance of length measurement, defined as the ratio of standard error to mean value, is as low as 3% between devices. The limit of detection (LOD) of our system is 600 fmol for AP and 400 fmol for αBCP. Using appropriate calibrations, we determined that an individual Aplysia bag cell neuron secretes 0.15 ± 0.03 pmol of AP and 0.13 ± 0.06 pmol of αBCP after being stimulated with elevated KCl. This quantitation approach is robust, does not require labeling, and is well suited for miniaturized off-line characterization from microfluidic devices.     

Cell-based high content screening using an integrated microfluidic device

High content screening (HCS) has quickly established itself as a core technique in the early stage of drug discovery for secondary compound screening. It allows several independent cellular parameters to be measured in a single cell or populations of cells in a single assay. In this work, we describe high content screening for the multiparametric measurement of cellular responses in human liver carcinoma (HepG2) cells using an integrated microfluidic device. This device consists of multiple drug gradient generators and parallel cell culture chambers, in which the processes of liquid dilution and diffusion, micro-scale cell culture, cell stimulation and cell labeling can be integrated into a single device. The simple assay provides multiparametric measurements of plasma membrane permeability, nuclear size, mitochondrial transmembrane potential and intracellular redox states in anti-cancer drug-induced apoptosis of HepG2 cells. The established platform is able to rapidly extract the maximum of information from tumor cells in response to several drugs varying in concentration, with minimal sample and less time, which is very useful for basic biomedical research and cancer treatment.     

Gene transfer and protein dynamics in stem cells using single cell electroporation in a microfluidic device

There is great interest in genetic modification of bone marrow-derived mesenchymal stem cells (MSC), not only for research purposes but also for use in (autologous) patient-derived-patient-used transplantations. A major drawback of bulk methods for genetic modifications of (stem) cells, like bulk-electroporation, is its limited yield of DNA transfection (typically then 10%). This is even more limited when cells are present at very low numbers, as is the case for stem cells. Here we present an alternative technology to transfect cells with high efficiency (>75%), based on single cell electroporation in a microfluidic device. In a first experiment we show that we can successfully transport propidium iodide (PI) into single mouse myoblastic C2C12 cells. Subsequently, we show the use of this microfluidic device to perform successful electroporation of single mouse myoblastic C2C12 cells and single human MSC with vector DNA encoding a green fluorescent-erk1 fusion protein (EGFP-ERK1 (MAPK3)). Finally, we performed electroporation in combination with live imaging of protein expression and dynamics in response to extracellular stimuli, by fibroblast growth factor (FGF-2). We observed nuclear translocation of EGFP-ERK1 in both cell types within 15 min after FGF-2 stimulation. Due to the successful and promising results, we predict that microfluidic devices can be used for highly efficient small-scale 'genetic modification' of cells, and biological experimentation, offering possibilities to study cellular processes at the single cell level. Future applications might be small-scale production of cells for therapeutic application under controlled conditions.     

Metabolic monitoring of the electrically stimulated single heart cell within a microfluidic platform

A device based on five individually addressable microelectrodes, fully integrated within a microfluidic system, has been fabricated to enable the real-time measurement of ionic and metabolic fluxes from electrically active, beating single heart cells. The electrode array comprised one pair of pacing microelectrodes, used for field-stimulation of the cell, and three other microelectrodes, configured as an electrochemical lactate microbiosensor, that were used to measure the amounts of lactate produced by the heart cell. The device also allowed simultaneous in-situ microscopy, enabling optical measurements of cell contractility and fluorescence measurements of extracellular pH and cellular Ca2+. Initial experiments aimed to create a metabolic profile of the beating heart cell, and results show well defined excitation-contraction (EC) coupling at different rates. Ca2+ transients and extracellular pH measurements were obtained from continually paced single myocytes, both as a function of the rate of cell contraction. Finally, the relative amounts of intra- and extra-cellular lactate produced during field stimulation were determined, using cell electroporation where necessary.     

A microfluidic processor for gene expression profiling of single human embryonic stem cells

The gene expression of human embryonic stem cells (hESC) is a critical aspect for understanding the normal and pathological development of human cells and tissues. Current bulk gene expression assays rely on RNA extracted from cell and tissue samples with various degree of cellular heterogeneity. These 'cell population averaging' data are difficult to interpret, especially for the purpose of understanding the regulatory relationship of genes in the earliest phases of development and differentiation of individual cells. Here, we report a microfluidic approach that can extract total mRNA from individual single-cells and synthesize cDNA on the same device with high mRNA-to-cDNA efficiency. This feature makes large-scale single-cell gene expression profiling possible. Using this microfluidic device, we measured the absolute numbers of mRNA molecules of three genes (B2M, Nodal and Fzd4) in a single hESC. Our results indicate that gene expression data measured from cDNA of a cell population is not a good representation of the expression levels in individual single cells. Within the G0/G1 phase pluripotent hESC population, some individual cells did not express all of the 3 interrogated genes in detectable levels. Consequently, the relative expression levels, which are broadly used in gene expression studies, are very different between measurements from population cDNA and single-cell cDNA. The results underscore the importance of discrete single-cell analysis, and the advantages of a microfluidic approach in stem cell gene expression studies.     

Microfluidic array cytometer based on refractive optical tweezers for parallel trapping, imaging and sorting of individual cells

Analysis of genetic and functional variability in populations of living cells requires experimental techniques capable of monitoring cellular processes such as cell signaling of many single cells in parallel while offering the possibility to sort interesting cell phenotypes for further investigations. Although flow cytometry is able to sequentially probe and sort thousands of cells per second, dynamic processes cannot be experimentally accessed on single cells due to the sub-second sampling time. Cellular dynamics can be measured by image cytometry of surface-immobilized cells, however, cell sorting is complicated under these conditions due to cell attachment. We here developed a cytometric tool based on refractive multiple optical tweezers combined with microfluidics and optical microscopy. We demonstrate contact-free immobilization of more than 200 yeast cells into a high-density array of optical traps in a microfluidic chip. The cell array could be moved to specific locations of the chip enabling us to expose in a controlled manner the cells to reagents and to analyze the responses of individual cells in a highly parallel format using fluorescence microscopy. We further established a method to sort single cells within the microfluidic device using an additional steerable optical trap. Ratiometric fluorescence imaging of intracellular pH of trapped yeast cells allowed us on the one hand to measure the effect of the trapping laser on the cells' viability and on the other hand to probe the dynamic response of the cells upon glucose sensing.     

Droplet microfluidics for amplification-free genetic detection of single cells

In this article we present a novel droplet microfluidic chip enabling amplification-free detection of single pathogenic cells. The device streamlines multiple functionalities to carry out sample digitization, cell lysis, probe-target hybridization for subsequent fluorescent detection. A peptide nucleic acid fluorescence resonance energy transfer probe (PNA beacon) is used to detect 16S rRNA present in pathogenic cells. Initially the sensitivity and quantification abilities of the platform are tested using a synthetic target mimicking the actual expression level of 16S rRNA in single cells. The capability of the device to perform "sample-to-answer" pathogen detection of single cells is demonstrated using E. coli as a model pathogen.     

Activated T lymphocytes migrate toward the cathode of DC electric fields in microfluidic devices

Immune cell migration is a fundamental process that enables immunosurveillance and immune responses. Understanding the mechanism of immune cell migration is not only of importance to the biology of cells, but also has high relevance to cell trafficking mediated physiological processes and diseases such as embryogenesis, wound healing, autoimmune diseases and cancers. In addition to the well-known chemical concentration gradient based guiding mechanism (i.e. chemotaxis), recent studies have shown that lymphocytes can respond to applied physiologically relevant direct current (DC) electric fields by migrating toward the cathode of the fields (i.e. electrotaxis) in both in vitro and in vivo settings. In the present study, we employed two microfluidic devices allowing controlled application of electric fields inside the microfluidic channel for quantitative studies of lymphocyte electrotaxis in vitro at the single cell level. The first device is fabricated by soft-lithography and the second device is made in glass with integrated on-chip electrodes. Using both devices, we for the first time showed that anti-CD3/CD28 antibodies activated human blood T cells migrate to the cathode of the applied DC electric field. This finding is consistent with previous electrotaxis studies on other lymphocyte subsets suggesting electrotaxis is a novel guiding mechanism for immune cell migration. Furthermore, the characteristics of electrotaxis and chemotaxis of activated T cells in PDMS microfluidic devices are compared.     

Characterization of a microfluidic dispensing system for localised stimulation of cellular networks

We present a 3-D microfluidic device designed for localized drug delivery to cellular networks. The device features a flow cell comprising a main channel for nutrient delivery as well as multiple channels for drug delivery. This device is one key component of a larger, fully integrated system now under development, based upon a microelectrode array (MEA) with on-chip CMOS circuitry for recording and stimulation of electrogenic cells (e.g. neurons, cardiomyocytes). As a critical system unit, the microfluidics must be carefully designed and characterized to ensure that candidate drugs are delivered to specific regions of the culture at known concentrations. Furthermore, microfluidic design and functionality is dictated by the size, geometry, and material/electrical characteristics of the CMOS MEA. Therefore, this paper reports on the design considerations and fabrication of the flow cell, including theoretical and experimental analysis of the mass transfer properties of the nutrient and drug flows, which are in good agreement with one another. To demonstrate proof of concept, the flow cell was mounted on a dummy CMOS chip, which had been plated with HL-1 cardiomyocytes. A test chemical compound was delivered to the cell culture in a spatially resolved manner. Envisioned applications of this stand-alone system include simultaneous toxicological testing of multiple compounds and chemical stimulation of natural neural networks for neuroscience investigations.